Genetically altering the disease vector status of insects using recombinant DNA technologies is being considered as an alternative to eradication eff

Eukaryotic genomes are organized into chromatin domains with three-dimensional arrangements that presumably result from interactions between the chromatin constituents—proteins, DNA, and RNA—within the physical constraints of the nucleus. We used chromosome conformation capture (3C) followed by high-throughput sequencing (Hi-C) with wild-type and mutant strains of Neurospora crassa to gain insight into the role of heterochromatin in the organization and function of the genome. We tested the role of three proteins thought to be important for establishment of heterochromatin, namely, the histone H3 lysine 9 methyltransferase DIM-5, Heterochromatin Protein 1 (HP1), which specifically binds to the product of DIM-5 (trimethylated H3 lysine 9 [H3K9me3]), and DIM-3 (importin alpha), which is involved in DIM-5 localization. The average genome configuration of the wild-type strain revealed strong intra- and inter-chromosomal associations between both constitutive and facultative heterochromatic domains, with the strongest interactions among the centromeres, subtelomeres, and interspersed heterochromatin. Surprisingly, loss of either H3K9me3 or HP1 had only mild effects on heterochromatin compaction, whereas dim-3 caused more drastic changes, specifically decreasing interactions between constitutive heterochromatic domains. Thus, associations between heterochromatic regions are a major component of the chromosome conformation in Neurospora, but two widely studied key heterochromatin proteins are not necessary, implying that undefined protein factors play key roles in maintaining overall chromosome organization.

Significance: Two forms of heterochromatin, constitutive and facultative, cause gene silencing in eukaryotes. In Neurospora crassa , H3K27me2/3-marked facultative heterochromatin reversibly represses scores of specialized genes, whereas H3K9me3-marked constitutive heterochromatin permanently silences repetitive DNA. Interactions between heterochromatin provide a structural framework for the genome, and this is thought to be functionally important. Histone marks underlying constitutive and facultative heterochromatin are nonessential in N. crassa , permitting tests of their roles in genome organization and gene expression. Although linkages between regions of constitutive heterochromatin are the most prominent feature of the 3D structure of the genome, loss of the facultative mark has a much greater effect on genome architecture than does loss of key features of constitutive heterochromatin, i.e., H3K9me3 and Heterochromatin Protein 1.

Summary: 6S RNA binds σ⁷⁰‐RNA polymerase and downregulates transcription at many σ⁷⁰‐dependent promoters, but others escape regulation even during stationary phase when the majority of the transcription machinery is bound by the RNA. We report that core promoter elements determine this promoter specificity; a weak −35 element allows a promoter to be 6S RNA sensitive, and an extended −10 element similarly determines 6S RNA inhibition except when a consensus −35 element is present. These two features together predicted that hundreds of mapped Escherichia coli promoters might be subject to 6S RNA dampening in stationary phase. Microarray analysis confirmed 6S RNA‐dependent downregulation of expression from 68% of the predicted genes, which corresponds to 49% of the expressed genes containing mapped E. coli promoters and establishes 6S RNA as a global regulator in stationary phase. We also demonstrate a critical role for region 4.2 of σ⁷⁰ in RNA polymerase interactions with 6S RNA. Region 4.2 binds the −35 element during transcription initiation; therefore we propose one mechanism for 6S RNA regulation of transcription is through competition for binding region 4.2 of σ⁷⁰.

Summary: 6S RNA is a small, non‐coding RNA that interacts with σ⁷⁰‐RNA polymerase and downregulates transcription at many promoters during stationary phase. When bound to σ⁷⁰‐RNA polymerase, 6S RNA is engaged in the active site of σ⁷⁰‐RNA polymerase in a manner similar enough to promoter DNA that the RNA can serve as a template for RNA synthesis. It has been proposed that 6S RNA mimics the conformation of DNA during transcription initiation, suggesting contacts between RNA polymerase and 6S RNA or DNA may be similar. Here we demonstrate that region 4.2 of σ⁷⁰ is critical for the interaction between 6S RNA and RNA polymerase. We define an expanded binding surface that encompasses positively charged residues throughout the recognition helix of the helix–turn–helix motif in region 4.2, in contrast to DNA binding that is largely focused on the N‐terminal region of this helix. Furthermore, negatively charged residues in region 4.2 weaken binding to 6S RNA but do not similarly affect DNA binding. We propose that the binding sites for promoter DNA and 6S RNA on region 4.2 of σ⁷⁰ are overlapping but distinct, raising interesting possibilities for how core promoter elements contribute to defining promoters that are sensitive to 6S RNA regulation.